Synthesis and biological evaluation of potential anti-cancer agents

The new technology of combinational chemistry has been introduced to pharmaceutical companies, improving and making more efficient the process of drug discovery. Automated combinatorial chemistry in the solution-phase has been used to prepare a large number of compounds of anti-cancer screening. A library of caffeic acid derivatives has been prepared by the Knoevenagel condensation of aldehyde and active methylene reagents. These products have been screened against two murine adenocarcinoma cell lines (MAC) which are generally refractive to standard cytotoxic agents. The target of anti-proliferative action was the 12- and 15-lipoxygenase enzymes upon which these tumour cell lines have been shown to be dependent for proliferation and metastasis. Compounds were compared to a standard lipoxygenase inhibitor and if found to be active anti-proliferative agents were tested for their general cytotoxicity and lipoxygenase inhibition. A solid-phase bound catalyst, piperazinomethyl polystyrene, was devised and prepared for the improved generation of Knoevenagel condensation products. This piperazinomethyl polystyrene was compared to the traditional liquid catalyst, piperidine, and was found to reduce the amount of by-products formed during reaction and had the advantage of easy removal from the reaction. 13C NMR has been used to determine the E/Z stereochemistry of Knoevenagel condensation products. Soluble polymers have been prepared containing different building blocks pendant to the polymer backbone. Aldehyde building blocks incorporated into the polymer structure have been subjected to the Knoevenagel condensation. Cleavage of the resultant pendant molecules has proved that soluble linear polymers have the potential to generate combinatorial mixtures of known composition for biological testing. Novel catechol derivatives have been prepared by traditional solution-phase chemistry with the intention of transferring their synthesis to a solid-phase support. Catechol derivatives prepared were found to be active inhibitors of lipoxygenase. Soluble linear supports for the preparation of these active compounds were designed and tested. The aim was to develop a support suitable for the automated synthesis of libraries of catechol derivatives for biological screening.

Automated synthesis and evaluation of potential new anti-microbial agents

A series of N1-benzylideneheteroarylcarboxamidrazones was prepared in an automated fashion, and tested against Mycobacterium fortuitum in a rapid screen for antimycobacterial activity. Many of the compounds from this series were also tested against Mycobacterium tuberculosis, and the usefulness as M.fortuitum as a rapid, initial screen for anti-tubercular activity evaluated. Various deletions were made to the N1-benzylideneheteroarylcarboxamidrazone structure in order to establish the minimum structural requirements for activity. The N1-benzylideneheteroarylcarbox-amidrazones were then subjected to molecular modelling studies and their activities against M.fortuitum and M.tuberculosis were analysed using quantitative structure-analysis relationship (QSAR) techniques in the computational package TSAR (Oxford Molecular Ltd.). A set of equations predictive of antimycobacterial activity was hereby obtained. The series of N1-benzylidenehetero-arylcarboxamidrazones was also tested against a multidrug-resistant strain of Staphylococcus aureus (MRSA), followed by a panel of Gram-positive and Gram-negative bacteria, if activity was observed for MRSA. A set of antimycobacterial N1-benzylideneheteroarylcarboxamidrazones was hereby discovered, the best of which had MICs against m. fortuitum in the range 4-8μgml-1 and displayed 94% inhibition of M.tuberculosis at a concentration of 6.25μgml-1. The antimycobacterial activity of these compounds appeared to be specific, since the same compounds were shown to be inactive against other classes of organisms. Compounds which were found to be sufficiently active in any screen were also tested for their toxicity against human mononuclear leucocytes. Polyethylene glycol (PEG) was used as a soluble polymeric support for the synthesis of some fatty acid derivatives, containing an isoxazoline group, which may inhibit mycolic acid synthesis in mycobacteria. Both the PEG-bound products and the cleaved, isolated products themselves were tested against M.fortuitum and some low levels of antimycobacterial activity were observed, which may serve as lead compounds for further studies.

Formulation of topical non-steroidal anti-inflammatory agents

Reversed-pahse high-performance liquid chromatographic (HPLC) methods were developed for the assay of indomethacin, its decomposition products, ibuprofen and its (tetrahydro-2-furanyl)methyl-, (tetrahydro-2-(2H)pyranyl)methyl- and cyclohexylmethyl esters. The development and application of these HPLC systems were studied. A number of physico-chemical parameters that affect percutaneous absorption were investigated. The pKa values of indomethacin and ibuprofen were determined using the solubility method. Potentiometric titration and the Taft equation were also used for ibuprofen. The incorporation of ethanol or propylene glycol in the solvent resulted in an improvement in the aqueous solubility of these compounds. The partition coefficients were evaluated in order to establish the affinity of these drugs towards the stratum corneum. The stability of indomethacin and of ibuprofen esters were investigated and the effect of temperature and pH on the decomposition rates were studied. The effect of cetyltrimethylammonium bromide on the alkaline degradation of indomethacin was also followed. In the presence of alcohol, indomethacin alcoholysis was observed and the kinetics of decomposition were subjected to non-linear regression analysis and the rate constants for the various pathways were quantified. The non-isothermal, sufactant non-isoconcentration and non-isopH degradation of indomethacin were investigated. The analysis of the data was undertaken using NONISO, a BASIC computer program. The degradation profiles obtained from both non-iso and iso-kinetic studies show that there is close concordance in the results. The metabolic biotransformation of ibuprofen esters was followed using esterases from hog liver and rat skin homogenates. The results showed that the esters were very labile under these conditions. The presence of propylene glycol affected the rates of enzymic hydrolysis of the ester. The hydrolysis is modelled using an equation involving the dielectric constant of the medium. The percutaneous absorption of indomethacin and of ibuprofen and its esters was followed from solutions using an in vitro excised human skin model. The absorption profiles followed first order kinetics. The diffusion process was related to their solubility and to the human skin/solvent partition coefficient. The percutaneous absorption of two ibuprofen esters from suspensions in 20% propylene glycol-water were also followed through rat skin with only ibuprofen being detected in the receiver phase. The sensitivity of ibuprofen esters to enzymic hydrolysis compared to the chemical hydrolysis may prove valuable in the formulation of topical delivery systems.

The biochemical pharmacology and toxicology of anti-parasitic agents

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In vitro toxicity and bioactivity of some pyrimethamine derivatives as anti-tuberculosis agents

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A bio-adhesive formulation for the delivery of anti-fungal agents to the oesophagus

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Total synthesis of furospongolide and related furanolipid analogues as potential anti-tumour agents

This thesis details the design, development and execution of innovative methodology in the total synthesis of the terpene-derived marine natural product, furospongolide. It also outlines the synthetic routes used to prepare a novel range of furanolipids derivatives and subsequent evaluation of their potential as antitumour agents. The first chapter is a review of the literature describing efforts undertaken towards the synthesis of biologically active furanosesterterpenoid marine natural products. A brief discussion on the sources and biological activity exhibited by furan natural products is also provided. In addition, a concise account of the role of hypoxia in cancer, and the increasing interest in HIF-1 inhibition as a target for chemotherapeutics is examined. The second chapter discusses the concise synthesis of the marine HIF-1 inhibitor furospongolide, which was achieved in five linear steps from (E,E)-farnesyl acetate. The synthetic strategy features a selective oxidation reaction, a Schlosser sp3-sp3 cross-coupling, a Wittig cross-coupling and an elaborate one-pot selective reduction, lactonisation and isomerization reaction to install the butenolide ring. The structure-activity relationship of furospongolide was also investigated. This involved the design and synthesis of a library of structurally modified analogues sharing the same C1-C13 subunit. This was achieved by exploiting the brevity and high level of convergence of our synthetic route together with the readily amenable structure of our target molecule. Exploiting the Schlosser cross-coupling allowed for replacement of furan with other heterocycles in the preparation of various furanolipid and thiophenolipid derivatives. The employment of reductive amination and Wittig chemistry further added to our novel library of structural derivatives. The third chapter discusses the results obtained from the NCI from biological evaluation From a collection of 28 novel compounds evaluated against the NCI-60 cancer cell array, six drug candidates were successfully selected for further biological evaluation on the basis of antitumour activity. COMPARE analysis revealed a strong correlation between some of our design analogues and the blockbuster anticancer agent tamoxifen, further supporting the potential of furanolipids in the treatment of breast cancer. The fourth chapter, details the full experimental procedures, including spectroscopic and analytical data for all the compounds prepared during this research.

Synthesis and evaluation of novel ellipticines as potential anti-cancer agents

Ellipticine is a natural product which possesses multimodal anti-cancer activity. This thesis encompasses the synthesis and biological evaluation of novel ellipticine and isoellipticine derivatives as anti-cancer agents. Expanding on previous work within the group utilising vinylmagnesium bromide, derivatisation of the C5 position of ellipticine was accomplished by reaction of a key ketolactam intermediate with Grignard reagents. Corresponding attempts to introduce diverse substitution at the C11 position were unsuccessful, although one novel C11 derivative was produced using an alkyllithium reagent. A panel of novel ellipticinium salts encompassing a range of substitutions at the N2, C9 and N6 positions were prepared. Extensive derivatisation of the N10 position of isoellipticine was undertaken for the first time. Novel substitution in the form of acid and methyl ester functionalities were introduced at the C7 position of isoellipticine while novel C7 aldehyde and alcohol derivatives were synthesised. A large panel of isoellipticinium salts were prepared with conditions adjusted for the reactivity of the alkyl halide. Novel coupling reactions to increase the yield of isoellipticine were attempted but proved unsuccessful. A panel of 54 novel derivatives was prepared and a multimodal analysis of their anti-cancer activity was conducted. The NCI 60-human tumour cell lines screen was a primary source of information on the in vitro activity of compounds with derivatives found to exert potent anticancer effects, with mean GI50 values as low as 1.01 μM across the full range of cancer types and as low as 16 nM in individual cell lines. A second in vitro screen in collaboration with researchers in the University of Nantes identified derivatives which could potently inhibit growth in a p53 mutant NSCLC cell line. The cell cycle effects of a selected panel of isoellipticines were studied in leukaemia cell lines by researchers in the Department of Biochemistry and Cell Biology, UCC. Emerging from this, the therapeutic potential of one of the derivatives in AML was then assessed in vivo in an AML xenograft mouse model, with tumour weight reduced by a factor of 7 in treated mice relative to control.

Anti-anxiety effect of methanol extract of Pericarpium zanthoxyli using a strychnine-sensitive glycine receptor model

Purpose: To determine if the methanol extract of Pericarpium zanthoxyli exerts anti-anxiety effects and also to explore any probable anti-anxiety mechanism in vivo. Methods: The staircase test, elevated plus maze test, rota-rod treadmill test and convulsions induced by strychnine and picrotoxin on mice were tested to identify potential mechanism of anti-anxiety activity of the plant extract. Results: The plant extract (10 mg/kg, p.o.) significantly reduced rearing numbers in the staircase test while it increased the time spent in the open arms as well as the number of entries to the open arms in the elevated plus maze test, suggesting that it has significant anti-anxiety activity. Furthermore, the extract inhibited strychnine-induced convulsion. However, it had little effect on picrotoxin-induced convulsion, suggesting that its anti-anxiety activity may be linked to strychnine-sensitive glycine receptor and not GABA receptor. Conclusion: These results suggest that the Pericarpium zanthoxyli extract may be beneficial for the control of anxiety.

Anti-inflammatory agents in the treatment of bipolar depression: a systematic review and meta-analysis

OBJECTIVE: Inflammation has been implicated in the risk, pathophysiology, and progression of mood disorders and, as such, has become a target of interest in the treatment of bipolar disorder (BD). Therefore, the objective of the current qualitative and quantitative review was to determine the overall antidepressant effect of adjunctive anti-inflammatory agents in the treatment of bipolar depression. METHODS: Completed and ongoing clinical trials of anti-inflammatory agents for BD published prior to 15 May 15 2015 were identified through searching the PubMed, Embase, PsychINFO, and Clinicaltrials.gov databases. Data from randomized controlled trials (RCTs) assessing the antidepressant effect of adjunctive mechanistically diverse anti-inflammatory agents were pooled to determine standard mean differences (SMDs) compared with standard therapy alone. RESULTS: Ten RCTs were identified for qualitative review. Eight RCTs (n = 312) assessing adjunctive nonsteroidal anti-inflammatory drugs (n = 53), omega-3 polyunsaturated fatty acids (n = 140), N-acetylcysteine (n = 76), and pioglitazone (n = 44) in the treatment of BD met the inclusion criteria for quantitative analysis. The overall effect size of adjunctive anti-inflammatory agents on depressive symptoms was -0.40 (95% confidence interval -0.14 to -0.65, p = 0.002), indicative of a moderate and statistically significant antidepressant effect. The heterogeneity of the pooled sample was low (I² = 14%, p = 0.32). No manic/hypomanic induction or significant treatment-emergent adverse events were reported. CONCLUSIONS: Overall, a moderate antidepressant effect was observed for adjunctive anti-inflammatory agents compared with conventional therapy alone in the treatment of bipolar depression. The small number of studies, diversity of agents, and small sample sizes limited interpretation of the current analysis.

Therapeutic response of benzodiazepine, orphenadrine citrate and occlusal splint association in TMD pain

Loss of function, muscle inflammation, and pain are some of the signs and symptoms of temporomandibular dysfunction (TMD). Pharmacological strategies to minimize the clinical manifestation of these disorders often focus on blocking or inhibiting the pain-causing symptom. Resources such as muscle-relaxants, anxiety-relief drugs, and splint therapy are often used to reduce muscular hyperactivity related to TMD muscle pain. This study compares the effect of a randomly ordered association of occlusal splint therapy (S), nonsteroid anti-inflammatory with a muscle-relaxant drug (orphenadrine citrate) (O), and an anxiety-relief drug (benzodiazepine) (B), to ease painful TMD muscle symptoms. Clinical and anamnestic analyses were recorded in accordance with the Helkimo TMD index and applied before and after treatments. Twenty-one group two Helkimo TMD adult female patients were treated, all of whom were subjected to the three random therapeutic associations proposed: SBO, BOS, and OSB. The same operator applied the three specific associations over a period of 21 days in the proposed sequence, seven days for each therapy. The results show that all the groups presented the best results in terms of relief from pain after the therapeutic association (28.5% showed a decrease and 47.6% showed an absence of symptoms). No significant difference was observed among association therapeutic protocols. Copyright © 2003 by CHROMA, Inc.

Citrus aurantium L. essential oil exhibits anxiolytic-like activity mediated by 5-HT1A-receptors and reduces cholesterol after repeated oral treatment

Background: The current treatments for anxiety disorders and depression have multiple adverse effects in addition to a delayed onset of action, which has prompted efforts to find new substances with potential activity in these disorders. Citrus aurantium was chosen based on ethnopharmacological data because traditional medicine refers to the Citrus genus as useful in diminishing the symptoms of anxiety or insomnia, and C. aurantium has more recently been proposed as an adjuvant for antidepressants. In the present work, we investigated the biological activity underlying the anxiolytic and antidepressant effects of C. aurantium essential oil (EO), the putative mechanism of the anxiolytic-like effect, and the neurochemical changes in specific brain structures of mice after acute treatment. We also monitored the mice for possible signs of toxicity after a 14-day treatment.Methods: The anxiolytic-like activity of the EO was investigated in a light/dark box, and the antidepressant activity was investigated in a forced swim test. Flumazenil, a competitive antagonist of benzodiazepine binding, and the selective 5-HT1A receptor antagonist WAY100635 were used in the experimental procedures to determine the mechanism of action of the EO. To exclude false positive results due to motor impairment, the mice were submitted to the rotarod test.Results: The data suggest that the anxiolytic-like activity observed in the light/dark box procedure after acute (5 mg/kg) or 14-day repeated (1 mg/kg/day) dosing was mediated by the serotonergic system (5-HT1A receptors). Acute treatment with the EO showed no activity in the forced swim test, which is sensitive to antidepressants. A neurochemical evaluation showed no alterations in neurotransmitter levels in the cortex, the striatum, the pons, and the hypothalamus. Furthermore, no locomotor impairment or signs of toxicity or biochemical changes, except a reduction in cholesterol levels, were observed after treatment with the EO.Conclusion: This work contributes to a better understanding of the biological activity of C. aurantium EO by characterizing the mechanism of action underlying its anxiolytic-like activity. © 2013 Costa et al; licensee BioMed Central Ltd.